n-Alkane profiles in rajma seed surface
The extraction of 100 g rajma seeds yielded 3.50 ± 0.012 mg of seed surface wax alkanes which is ca. 10% of the total crude extract of surface waxes. Table 1 and Fig. 1 shows 18
n-alkanes in the surface waxes of rajma seeds. Octacosane (
n-C
28) was the predominant
n-alkane, accounting for 846.67±17.64µg. Heneicosane (
n-C
21) was the second most abundant alkane followed by
n-tritriacontane (
n-C
33) in the surface waxes of rajma seeds.
n-Hexadecane (
n-C
16) and
n-pentadecane (
n-C
15) were the least abundant alkanes in rajma seeds. The rest 13
n-alkanes displayed different patterns in the surface waxes of rajma seeds.
Y-tube olfactometer bioassay
The results obtained in the series of olfactometric bioassays showing effectiveness of
C.chinensis towards alkanes isolated from
P. vulgaris L. seeds surface waxes are presented in Table 2. Alkanes from the rajma seed surface attracted the insect significantly at 0.5 seed equivalent (63.33%; χ2=6.4; df =1; P<0.05), 1 seed equivalent (68.89%; χ2=12.84; df =1; P< 0.001), 2 seeds equivalent (73.33%; χ2=19.6; df =1; P< 0.001), 4 seeds equivalent (81.11%; χ2=34.84; df =1; P<0.00001) and 6 seeds equivalent (86.67%; χ2=48.4; df =1; P<0.00001). Insects’ did not respond towards 0.25 seed equivalent.
Bioassays with the mixtures of synthetic alkanes mimicking the surface wax alkanes of
P. vulgaris L. seeds are also summarized in Table 2. Insects showed attraction towards a mimic of 0.5 seed equivalent (61.11%; χ2=4.4; df =1; P<0.05), 1 seed equivalent (66.67%; χ2=10; df =1; P< 0.01), 2 seeds equivalent (70%; χ2=14.4; df =1; P< 0.001), 4 seeds equivalent (78.89%; χ2=30.04; df =1; P<0.00001) and 6 seeds equivalent (84.44%; χ2=42.71; df =1; P<0.00001).
Table 3 represents the results of olfactory bioassays of
C.chinensis for individual synthetic alkanes mimicking the surface wax alkanes of
P. vulgaris L. seeds. The insect responded positively to
n-C
21 at 1.16 µg (58.89%; χ2=4.4; df =1; P=0.09), 2.32 µg (63.33%; χ2=6.4; df =1; P=0.011), 4.64µg (64.44%; χ2=7.51; df =1; P<0.01), 9.28 µg (67.78%; χ2=11.38; df =1; P<0.001) and 13.92 µg (70%; χ2=14.4; df =1; P<0.001). Females also showed positive responses towards
n-C
25 at 0.569 µg (58.89%; χ2=4.4; df =1; P=0.09), 1.138 µg (64.44%; χ2=7.51; df =1; P<0.01), 2.276 µg (66.67%; χ2=10; df =1; P=0.001), 4.552 µg (67.78%; χ2=11.38; df =1; P<0.001) and 6.828 µg (70%; χ2=14.4; df =1; P<0.001). A positive response was also recorded for
n-C
15 at 0.564 µg (62.22%; χ2=5.37; df =1; P=0.02) and the response for the same was the highest at 1.692 µg (66.67%; χ2=10; df =1; P=0.001). Females showed attractions towards
n-C
16 at 1.938 µg (60%; χ2=3.6; df =1; P=0.057),
n-C
20 at 1.692 µg (56.67%; χ2=1.6; df =1; P=0.2),
n-C
22 at 2.94 µg (60%; χ2=3.6; df =1; P=0.057) and 4.41 µg (62.22%; χ2=5.37; df =1; P=0.02),
n-C
24 at 4.92 µg (61.11%; χ2=4.4; df =1; P<0.05),
n-C31 at 4.72 µg (61.11%; χ2=4.4; df =1; P<0.05) and
n-C
32 at 1.17 µg (63.33%; χ2=6.4; df =1; P=0.011) and 1.76 µg (67.78%; χ2=11.38; df =1; P<0.001). Rest of the alkanes did not show attraction in the bioassays. A combination of nine alkane’s mixture mimicking the amounts present in the seed surface waxes showed 65.56, 68.89, 76.67 and 80% attraction at 1 seed equivalent (χ2=8.71; df =1; P<0.01), 2 seeds equivalent (χ2=12.84; df =1; P<0.001), 4 seeds equivalent (χ2=25.6; df =1; P<0.00001) and 6 seeds equivalent (χ2=32.4; df =1; P<0.00001), respectively.
Alkanes are the most common component in plant and seed surface waxes. They have different roles in plant insect interactions such as attractants for feeding (
Dutton et al.,2000;
Tasin et al.,2005;
Sarkar et al.,2013a;
Mukherjee et al.,2013;
Sarkar and Barik, 2014;
Mitra et al.,2019) or stimulants for oviposition (
Eigenbrode and Espelie, 1995;
Li and Ishikawa, 2006;
Das et al.,2019;
Mitra et al.,2020). A study by
Parr et al.,(1998) on chickpea seed surface wax showed that heptacosane (
n-C
27) and nonacosane (
n-C
29) were the most abundant
n-alkanes.
n-Alkanes with chain lengths from
n-C
15 to
n-C
32 were present in the surface waxes of khesari seeds among four variety. Further,
n-C19 was the most predominant alkane in surface waxes of four varities of khesari seeds
(Adhikary et al., 2014). However in our study,
n-C
28 was the most predominant alkane in the surface waxes of rajma seeds.
The olfactory bioassay study provides evidence that the long-chain alkanes can act as close range attractant for
C.chinensis. Different studies by previous authors demonstrated the importance of surface wax alkanes in different insects (
Schiestl et al.,1999;
Tasin et al.,2005;
Roy and Barik, 2012;
Mukherjee et al.,2013;
Sarkar et al.,2013a;
Adhikary et al.,2014;
Mitra et al.,2020).
Kotze et al.,(2010) showed that alkanes from flowers of
Acacia cyclops A. Cunn.ex G. Don are used by a gall midge,
Dasineura dielsi Rübsaamen for finding its host.
Aulacophora foveicollis Lucas females elicited attraction to a synthetic blend of
n-C
19,
n-C
27 and
n-C
29 alkanes mimicking the amounts present in 6 mg surface wax alkanes of
Momordicacochin chinensis Spreng flowers (
Mukherjee et al.,2013).
Adhikary et al.,(2014) showed that a blend of
n-C
15,
n-C
18,
n-C
19,
n-C
21,
n-C
23 and
n-C
25 with the amount of 0.33, 0.20, 1.16, 0.94, 0.75 and 0.56 µg, respectively attracted the
C. maculatus.
Aphis craccivora females showed attraction towards a synthetic blend of pentadecane, octadecane, docosane, pentacosane, heptacosane, octacosane, nonacosane resembling the amounts present in the leaf surface waxes of BIO L 212 Ratan (BIO) and Nirmal B-1 (NIR) cultivars of
Lathyrus sativus (
Mitra et al.,2020)
. In the present study, a clear positive attraction (P<0.01) of the insect was recorded at 0.5 seed equivalent surface wax alkanes from rajma seeds, but the highest attraction was recorded (P < 0.00001) at 6 seeds equivalent. In general, our results provide evidences that
C.chinensis, is highly attracted towards 6 seeds equivalent surface wax alkane of rajma seeds and also a synthetic blend of alkane mixture produced same response (P<0.00001) as 6 seeds equivalent surface wax alkanes. We also identified the specific alkanes and their quantity in the surface wax of rajma seeds and suggest that a synthetic blend of nine alkanes (
n-C
15,
n-C
16,
n-C
20,
n-C
21,
n-C
22,
n-C
24,
n-C
25,
n-C
31 and
n-C
32) mimicking 6 seeds equivalent may be applied in lures to develop baited trap to control the outbreak of this insect. Different oviposition
-deterrent and toxic effects of various botanicals like
Azadirachta indica, Milletiaie ferrnginea and
Chrysanthemum cineraraefolium oil had been already known (
Mulatu and Gebremedhin, 2000) but no such compounds are available, which can be used as baited trap to control this insect pest. However, further experiment is needed to evaluate the response of the blend combined with those nine synthetic alkanes towards
C.chinensis in storage condition.